(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/096923 Al 23 June 2016 (23.06.2016) W P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/82 (2006.01) C12Q 1/68 (2006.01) kind of national protection available): AE, AG, AL, AM, C12N 15/113 (2010.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) Number: International Application DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/EP20 15/079893 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, 15 December 2015 (15. 12.2015) MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (25) Filing Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (26) Publication Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 14307040.7 15 December 2014 (15. 12.2014) EP kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicants: PARIS SCIENCES ET LETTRES - TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, QUARTIER LATIN [FR/FR]; 62bis, rue Gay-Lussac, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, 75005 Paris (FR). INSTITUT NATIONAL DE LA DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, SANTE ET DE LA RECHERCHE MEDICALE (IN- LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SERM) [FR/FR]; 101, rue de Tolbiac, 75013 Paris (FR). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, CENTRE NATIONAL DE LA RECHERCHE SCIEN- GW, KM, ML, MR, NE, SN, TD, TG). TIFIQUE (CNRS) [FR/FR]; 3, rue Michel Ange, 75016 Paris (FR). Published: — with international search report (Art. 21(3)) (72) Inventors: NAVARRO, Lionel; 12, allee des Platanes, 92160 Antony (FR). THIEBEAULD, Odon; 2, rue Brun, — with sequence listing part of description (Rule 5.2(a)) 92340 Bourg La Reine (FR). (74) Agent: REGIMBEAU; 20, rue de Chazelles, 75847 Paris Cedex 17 (FR). (54) Title: METHODS TO MONITOR POST-TRANSCRIPTIONAL GENE SILENCING ACTIVITY IN PLANT TISSUES/CELL © TYPES RELEVANT FOR PATHOGEN ENTRY, PROPAGATION OR REPLICATION 2 7) Abstract: The invention relates to transgenic plants comprising an inverted-repeat construct which triggers post-transcriptional gene silencing of an endogenous visual reporter gene driven by a tissue-specific promoter wherein said tissue is relevant for patho - gen entry, propagation or replication and their uses for screening natural or synthetic molecules, microorganisms or extracts from mi- cro- or macro -organisms for their potential ability to inhibit pathogen entry, propagation or replication in plants by enhancing PTGS or for characterizing the mode of action of natural or synthetic molecules that are known to enhance plant disease resistance through an ill-defined mode of action. METHODS TO MONITOR POST-TRANSCRIPTIONAL GENE SILENCING ACTIVITY IN PLANT TISSUES/CELL TYPES RELEVANT FOR PATHOGEN ENTRY, PROPAGATION OR REPLICATION The present invention pertains to the field of agriculture. The invention relates to a method to monitor post-transcriptional gene silencing (PTGS) activity triggered by inverted-repeat in plant tissues/cell types that are relevant for the entry, propagation or replication of phytopathogens. These reporter systems can for instance be used to characterize the modes of action of known natural/synthetic compounds, microorganisms or extracts from micro- or macro-organisms that are known to promote disease resistance in crops. In addition, these reporter lines can be used to identify novel natural/synthetic compounds, microorganisms or extracts from micro- or macro-organisms that can promote PTGS and likely increase disease resistance in various cultivated plants. BACKGROUND AND STATE OF THE ART The innate immune response is the first line of defense against pathogens, and plays a critical role in antimicrobial defence. This response is initiated by host-encoded Pattern-Recognition Receptors (PRRs) that recognize evolutionarily conserved pathogen-derived signatures, known as Microbe-Associated Molecular Patterns (MAMPs), and activate MAMP-triggered immunity (MTI) (Boiler & Felix, 2009). Furthermore, plants have evolved another strategy to perceive microbial pathogens through disease resistance (R) proteins, which recognize, directly or indirectly, divergent pathogen virulence determinants known as effector proteins, and establish effector-triggered immunity (ETI) (Jones & Dangl, 2006). Upon detection of MAMPs or pathogen effectors, plant cells rapidly induce a series of signalling events that involve for instance, the differential expression of short interfering RNAs (siRNAs) and microRNAs (miRNAs) (Pumplin N & Voinnet O, 2013). Recently, several siRNAs and miRNAs were found to orchestrate MTI and ETI responses (Weiberg et a , 2014), implying a key role of RNA silencing in the regulation of the plant immune system. RNA silencing is an ancestral gene regulatory mechanism that controls gene expression at the transcriptional (TGS, Transcriptional Gene Silencing) and post-transcriptional (PTGS, Post- transcriptional Gene Silencing) levels. In plants, this mechanism has been initially characterized in antiviral resistance (Hamilton & Baulcombe, 1999). The core mechanism of RNA silencing starts with the production of double stranded RNAs (dsRNAs) that are processed by RNase-III enzymes DICERs into 20-24 nt small RNA duplexes that subsequently associate with an Argonaute (AGO) protein, which represent the central component of the RNA-induced silencing complexes (RISC). One strand, the guide, remains bound to the AGO effector protein to regulate genes in a mature RISC, while the other strand, the passenger, is degraded. The plant model Arabidopsis thaliana encodes 4 DICER-like proteins and 10 AGOs. DCL1 processes miRNA precursors into miRNA/miRNA* duplexes and the guide miRNA strand directs AGO 1-RISC to sequence complementary mRNA targets to trigger their degradation and/or translation inhibition. DCL2 and DCL4 are involved in the biogenesis of short interfering RNAs (siRNAs) derived from viral dsRNAs and play a critical role in antiviral resistance (Deleris et al, 2006; Diaz-Pendon et al., 2007). These DICER-like proteins are also involved, together with DCL3, in the production of siRNAs derived from transposable elements, read through, convergent or overlapping transcription, endogenous hairpins as well as some miRNA precursors (Bologna & Voinnet, 2014). In addition, a large proportion of dsRNAs are produced by RNA-dependent RNA polymerases (RDRs) that convert single stranded RNAs into dsRNAs. RDR6, which is one out of six Arabidopsis RDRs, produces dsRNAs from viral, transgene transcripts as well as some endogenous transcripts including transposable elements (Mourrain et al, 2000; Dalmay et al, 2000.; Schwach et al, 2005; Xie et al, 2004). These dsRNAs are processed in part by DCL4 and DCL2 into 2 1 nt and 22 nt siRNAs, respectively, which direct PTGS of endogenous sequence complementary mRNA targets or exogenous RNAs derived from sense-transgenes or viral RNAs (Bologna & Voinnet, 2014). Furthermore, both siRNA and miRNA duplexes are methylated by the small RNA methyltransferase HEN1 and this modification is essential for their stability (Yu et al, 2005; Li et al, 2005; Chen et al, 2012). Although endogenous miRNAs and siRNAs were initially characterized in various plant development processes, they have more recently emerged as key regulators of the plant innate immune response (Pumplin & Voinnet, 2013). For instance, the miR393 is a conserved microRNA that is induced during MTI and that contributes to antibacterial resistance in Arabidopsis (Navarro et al, 2006, 2008; Fahlgren et al, 2007; Li et al, 2010). Furthermore, phenotypical analyses in mutants that are defective in PTGS exhibit enhanced disease susceptibility to fungal, bacterial and oomycete pathogens (Navarro et al, 2008; Qiao et al, 2013; Ellendorff U et al, 2009; Navarro & Voinnet, 2008 WO/2008/087562), supporting a central role of this gene regulatory process in resistance against unrelated pathogens. However, despite the well-established role of RNA silencing in resistance against viral and non-viral pathogens, very little is known on the physiological relevance, or on the activity, of R A silencing in tissues that are relevant for the entry and/or propagation of phytopathogens. Phytopathogenic microbes can be divided into biotrophs, hemibiotrophs and necrotrophs according to their different lifestyles. Biotrophic pathogens can take-up nutrients from living host cells and maintain host cell viability, while necrotrophs kill host cells and feed on dead tissues. Hemibiotrophs use an early biotrophic phase followed by a necrotrophic phase. These phytopathogenic microbes use different strategies to enter inside host tissues. The majority of fungal and oomycete pathogens first produce spores, which adhere to the plant surface and further germinate to form a germ tube. Subsequently, the germ tube develops an appressorium that can perforate, through a penetration peg, the cuticle and cell wall layer through mechanical forces (Horbach et al., 201 1) and therefore enter inside host tissues. Once inside plant tissues, the hyphal tip forms a second specialized structure referred to as the haustorium that can uptake nutrients from host cells but also represents a major site of pathogen effector secretion (Mendgen & Hahn, 2002; Horbach et al., 201 1). Other pathogens do not perforate the cuticle cell wall layer but instead use natural openings to reach internal host tissues. For instance, hemibiotrophic bacterial pathogens such as Pseudomonas and Xanthomonas use hydathodes, stomata or woundings as natural entry sites for their endophytic colonization (Dou & Zhou, 2012). These bacterial pathogens can also enter plant tissues through the base of trichomes in some instances (Xin & He, 2013).